U.S. patent number 5,192,772 [Application Number 07/747,454] was granted by the patent office on 1993-03-09 for therapeutic agents.
This patent grant is currently assigned to Nippon Shinyaku Co. Ltd.. Invention is credited to Kazuya Mori, Nobutoshi Ojima, Yoshiaki Yoshikuni.
United States Patent |
5,192,772 |
Yoshikuni , et al. |
March 9, 1993 |
**Please see images for:
( Certificate of Correction ) ** |
Therapeutic agents
Abstract
Compounds are described which are useful for lowering the
activity of .alpha..sub.2 -PI, for the treatment of myocardial
infarctions and cerebral infarctions, as urokinase secretion
accelerators, as antithrombotic agents and to accelerate the
fibrinolysis accelerating effect.
Inventors: |
Yoshikuni; Yoshiaki (Uji,
JP), Ojima; Nobutoshi (Moriyama, JP), Mori;
Kazuya (Kyoto, JP) |
Assignee: |
Nippon Shinyaku Co. Ltd.
(JP)
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Family
ID: |
27339166 |
Appl.
No.: |
07/747,454 |
Filed: |
August 12, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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281495 |
Dec 8, 1988 |
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Foreign Application Priority Data
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Dec 9, 1987 [JP] |
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62-311348 |
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Current U.S.
Class: |
514/315; 514/317;
514/318; 514/319; 514/321; 514/326; 514/328 |
Current CPC
Class: |
A61K
31/435 (20130101); A61K 31/445 (20130101); A61K
31/70 (20130101); A61K 31/7004 (20130101) |
Current International
Class: |
A61K
31/70 (20060101); A61K 31/435 (20060101); A61K
31/445 (20060101); A61K 031/445 () |
Field of
Search: |
;514/230.5,315,321,318,326,328,319,317 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0034784 |
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Sep 1981 |
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EP |
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202661 |
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Nov 1986 |
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EP |
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295538 |
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Dec 1988 |
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EP |
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2067989 |
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Jan 1984 |
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GB |
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8703903 |
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Jul 1987 |
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WO |
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Other References
Blomback et al, Plasma Proteins, 1976, pp. 288-308. .
Aoki and Harpel, Seminars in Thrombosis and Hemostasis--vol. 10,
No. 1, 1984. .
Dictionary of Organic Compounds, 5th Edition, 2nd Supplement.
Chapman & Hall, publication 1984..
|
Primary Examiner: Waddell; Frederick E.
Assistant Examiner: Weddington; K.
Attorney, Agent or Firm: Rosenman & Colin
Parent Case Text
This application is a continuation of Ser. No. 07/281,495 filed
Dec. 8, 1988, now abandoned.
Claims
What is claimed is:
1. A method of accelerating fibronoylsis in humans and animals
which comprises administering to a human or animal in need thereof
an effective amount sufficient to accelerate fibronolysis of a
compound of the formula: ##STR3## a pharmaceutically acceptable
acid addition salt thereof or a quaternary salt thereof wherein
R.sup.2 is hydrogen, alkyl of 1 to 10 carbon atoms, carboxyalkyl of
1 to 10 carbon atoms in the alkyl moiety, lower
alkyloxycarbonylalkyl of 1 to 10 carbon atoms int he alkyl moiety,
hydroxyalkyl of 1 to 10 carbon atoms, cycloalkyl lower alkyl of 3
to 7 carbon atoms in the cycloalkyl moiety, arylalkyl of 1 to 10
carbon atoms in the alkyl moiety, aryloxyalkyl of 1 to 10 carbon
atoms in the alkyl moiety, alkenyl of 2 to 10 carbon atoms,
hydroxyalkenyl of 2 to 10 carbon atoms, arylalkenyl of 2 to 10
carbon atoms in the alkenyl moiety, aryloxyalkenyl of 2 to 10
carbon atoms in the alkenyl moiety or lower alkylcarbamoylalkyl of
1 to 10 carbon atoms int he alkyl moiety in combination with a
pharmaceutically acceptable carrier.
2. A method according to claim 1 wherein R.sup.2 is hydrogen, lower
alkyl, carboxy lower alkyl, lower alkyloxycarbonyl lower alkyl,
hydroxy lower alkyl, cycloalkyl lower alkyl of 3 to 5 carbon atoms
in the cycloalkyl moiety, aryl lower alkyl, aryloxy lower alkyl,
lower alkenyl, hydroxy lower alkenyl, aryl lower alkenyl, aryloxy
lower alkenyl, or lower alkylcarbamoyl lower alkyl wherein the aryl
moiety is phenyl or naphthyl.
Description
The present invention relates to compounds useful as .alpha..sub.2
-plasmin inhibitor depressants, urokinase secretion accelerators,
antithrombotic agents, accelerators for accelerating the
fibrinolysis accelerating effect and for the treatment of
myocardial and cerebral infarctions in humans and animals.
It is known in the art that for the formation of hemostatic
thromboses caused by physical damage to the blood vessels,
aggregation of platelets and subsequent precipitation of fibrin are
inevitably required. On the other hand, thromboses inhibit blood
flow in the blood vessels thus causing ischemia or necrosis of
tissue resulting in myocardial infarctions or cerebral infarctions.
Thus, the living body is prepared with a mechanism for removing
excess thromboses in the blood vessels where fibrinolysis by the
plasminogen-plasmin system plays a major role in the mechanism.
It is also known in the art that plasminogen is activated with a
plasminogen activator for conversion into plasmin and that plasmin
decomposes fibrin (fibrinolysis). An abnormality in this mechanism
would cause diseases such as myocardial or cerebral
infarctions.
One method of treating such conditions involve fibrinolytic therapy
in which the formed thrombus is lysed to improve the ischemic state
of the tissue. This is achieved by administering plasminogen
activators such as urokinase, streptokinase and the like. Thus, a
substance which accelerates the secretion of urokinase would be a
useful drug in the therapy of thromboses.
The living body is also prepared with a mechanism for protecting
against excessive fibrinoloysis. It is known, for example, that
.alpha..sub.2 -plasmin inhibitors (hereinafter referred to as
".alpha..sub.2 -PI") which is a plasmin inhibitor which
instantaneously inhibits plasmin activity thus inhibiting
fibrinoloysis. Therefore, .alpha..sub.2 -PI acts as a treatment
inhibitor in the therapy of fibrinoloysis and is also formed as a
type of protein in the acute phase to become one of the causes of
postoperative thromboses.
Thus, a drug which lowers the activity of an .alpha..sub.2 -PI
would enhance the effect in therapy of fibrinolysis and would
prevent postoperative thromboses from forming.
Thus, one of the objects of the present invention is to provide an
.alpha..sub.2 -PI inhibitor for administration for the treatment of
myocardial infarctions or cerebral infarctions.
A further object of the present invention is to provide a substance
for accelerating the secretion of urokinase since urokinase is a
plasminogen activator.
It has now been discovered that a compound selected from the group
consisting of:
a. a compound of the formula ##STR1## , a pharmaceutically
acceptable ac addition salt thereof or a quaternary salt thereof
wherein R is hydrogen, alkyl of 1 to 10 carbon atoms, carboxyalkyl
of 1 to 10 carbon atoms in the alkyl moiety, lower
alkyloxycarbonylalkyl of 1 to 10 carbon atoms in the alkyl moiety,
hydroxyalkyl of 1 to 10 carbon atoms in the alkyl moiety,
cycloalkyl lower alkyl wherein the cycloalkyl moiety is of 3 to 7
carbon atoms, aryl lower alkyl, aryloxy lower alkyl, alkenyl of 2
to 10 carbon atoms or arylalkenyl of 2 to 10 carbon atoms in the
alkenyl moiety;
b. nojirimycin or a pharmaceutically acceptable acid addition salt
thereof;
c. 1,4-bis(3-moranolino-1-propenyl)benzene or a pharmaceutically
acceptable acid addition salt thereof; and
d. castanospermine, are useful for lowering the activity of
.alpha..sub.2 -PI. Those compounds are also useful, therefore, in
the treatment of myocardial and cerebral infarctions and for the
treatment of thrombosis in humans and animals.
A compound selected from the group consisting of:
a. a compound of the formula ##STR2## or a pharmaceutically
acceptable acid addition salt thereof or a quaternary salt thereof
wherein R.sup.2 is hydrogen, alkyl of 1 to 10 carbon atoms,
carboxyalkyl of 1 to 10 carbon atoms in the alkyl moiety, lower
alkyloxycarbonylalkyl of 1 to 10 carbon atoms in the alkyl moiety,
hydroxyalkyl of 1 to 10 carbon atoms, cycloalkyl lower alkyl of 3
to 7 carbon atoms in the cycloalkyl moiety, arylalkyl of 1 to 10
carbon atoms in the alkyl moiety, aryloxyalkyl of 1 to 10 carbon
atoms in the alkyl moiety, alkenyl of 2 to 10 carbon atoms,
hydroxyalkenyl of 2 to 10 carbon atoms, arylalkenyl of 2 to 10
carbon atoms in the alkenyl moiety, aryloxyalkenyl of 2 to 10
carbon atoms in the alkenyl moiety or lower alkylcarbamoylalkyl of
1 to 10 carbon atoms in the alkyl moiety;
b. nojirimycin or a pharmaceutically acceptable acid addition salt
thereof;
c. 1,4-bis(3-moranolino-1-propenyl)benzene or a pharmaceutically
acceptable acid addition salt thereof; and
d. castanospermine, are useful as urokinase secretion accelerators
and for accelerating the fibrinoloysis accelerating effect.
The term alkyl as used herein refers to an alkyl moiety having 1 to
10 carbon atoms, preferably, lower alkyl. Representative lower
alkyl moieties are those containing from 1 to 6 carbon atoms such
as methyl, ethyl, propyl, butyl and the like.
Carboxyalkyl groups include those containing from 1 to 10 carbon
atoms in the alkyl moiety, but those having a lower alkyl moiety
are preferred. Representative carboxyalkyl moieties include
carboxymethyl, carboxyethyl and carboxypropyl.
Alkyloxycarbonylalkyl includes those moieties having 1 to 10 carbon
atoms in each of the alkyl moieties. It is preferred, however, that
each of the alkyl moieties be a lower alkyl moiety. Representative
moieties include methoxycarbonylmethyl, methoxycarbonylethyl,
methooxycarbonylpropyl, methoxycarbonylbutyl, ethoxycarbonylmethyl,
ethoxycarbonylethyl, ethoxycarbonylpropyl and
ethoxycarbonylbutyl.
The hydroxyalkyl moiety contains from 1 to 10 carbon atoms in the
alkyl moiety. Preferred hydroxyalkyl groups are hydroxy lower alkyl
moieties. Representative hydroxy lower alkyl moieties include
hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl.
Cycloalkyl alkyl moieties, preferably contain 3 to 7 carbon atoms
in the cycloalkyl moiety and 1 to 10 carbon atoms in the alkyl
moiety. Preferred groups include cycloalkyl alkyl moieties of 3 to
5 carbon atoms in the cycloalkyl moiety and wherein the alkyl
moiety is a lower alkyl group. Representative moieties include
cyclopropylmethyl, cyclobutylmethyl and cyclopentylmethyl.
Arylalkyl moieties contain from 1 to 10 carbon atoms in the alkyl
moiety, but preferably have lower alkyl moieties. The aryl moiety
is preferrably phenyl or naphthyl. Representative arylalkyl
moieties include benzyl, phenethyl, phenylpropyl, phenylbutyl,
phenylpentyl, naphthylmethyl, naphthylethyl, naphthylpropyl and
naphthylbutyl.
Aryloxyalkyl moieties are those of 1 to 10 carbon atoms in the
alkyl moiety, but preferably those wherein the aryl moeity is
phenyl or naphthyl and the alkyl moiety is a lower alkyl moiety.
Representative groups include phenoxymethyl, phenoxyethyl,
phenoxypropyl, phenoxybutyl, naphthyloxymethyl, naphthyloxyethyl,
naphthyloxypropyl and naphthyloxybutyl.
The alkenyl moieties contain from 2 to 10 carbon atoms; it is
preferred that they be lower alkenyl moieties. Representative
alkenyl moieties include vinyl, propenyl and butenyl.
Hydroxyalkenyl are those moieties of 2 to 10 carbon atoms in the
alkenyl moiety. Preferred hydroxyalkenyl moieties are hydroxy lower
alkenyl moieties. Representative groups include hydroxyvinyl,
hydroxypropenyl and hydroxybutenyl.
Arylalkenyl groups are those containing from 2 to 10 carbon atoms
in the alkenyl group, but preferably are those wherein the aryl
moiety is phenyl or naphthyl and the alkenyl moiety is a lower
alkenyl moiety. Representative groups include phenylvinyl,
phenylpropenyl, phenylbutenyl, naphthylvinyl, naphthylpropenyl and
naphthylbutenyl.
Aryloxyalkenyl moieties contain from 2 to 10 carbon atoms in the
alkenyl moiety, but preferably are those wherein the aryl moiety is
phenyl or naphthyl and the alkenyl moiety is a lower alkenyl
moiety.
Alkylcarbamoylalkyl moieties are those containing from 1 to 10
carbon atoms in each of the alkyl moieties, but preferably are
those wherein each of the alkyl moieties is a lower alkyl
group.
The arylalkyl, aryloxyalkyl and arylalkenyl moieties may optionally
be substituted.
In addition to the compounds described above, the present invention
also includes nojirimycin which has an OH group at 1-position of
the basic structure of moranoline and derivatives thereof, for
example, N-substituted derivatives. These compounds exhibit similar
activities to the moranoline derivatives, i.e., the unsubstituted
derivatives, and are included within the present invention.
Compounds having the moranoline basic structure as bis-form are
also included in the present invention. These compounds exhibit the
therapeutic utilities described above.
Representative examples of those compounds include nojirimicin and
pharmaceutically acceptable salts thereof such as
1,4-bis-(3-moranolino-1-propenyl)benzene and pharmaceutically
acceptable salts thereof. These compounds also exhibit an excellent
.alpha..sub.2 -PI lowering activity and urokinase secretion
accelerating activity.
The compounds of the present invention can be prepared by
conventional procedures per se known. The following nonlimitative
examples represent typical procedures for preparing
N-butylmoranoline.
PREPARATION EXAMPLE 1
Moranoline, 50 g, 126 g of n-butyl bromide and 170 g of potassium
carbonate were added to 1300 ml of dimethylformamide. The mixture
was stirred at room temperature for 7 days to complete the
reaction. After impurities were removed by filtration, the solvent
was distilled off under reduced pressure and 1000 ml of a strongly
acidic ion exchange resin Dowex 50W.times.2 (H.sup.+) was passed.
After thoroughly washing with water, elution was performed with 1N
ammonia water. The eluate was concentrated under reduced pressure.
Thereafter, 50 ml of methanol was added to the concentrate. The
mixture was allowed to stand at room temperature and the formed
crystals (47 g) were collected.
After the crystals were dissolved in 500 ml of methanol with
heating, the solution was cooled to room temperature and then
treated with activated charcoal. After concentrating to about 100
ml, the concentrate was allowed to stand at room temperature and 40
g of crystals precipitated were collected. After the crystals were
dissolved in 200 ml of methanol with heating, the solution was
gently concentrated. The concentrate was allowed to stand at room
temperature and crystals precipitated were collected. The crystals
were thoroughly dried at 70.degree. C. under reduced pressure to
give 34 g of objective N-(n-butyl)moranoline.
Yield, 50.6%; melting point, 128.degree.-129.degree. C.
Elemental analysis:
Calcd. (%) : C: 54.78; H: 9.65; N: 6.39,
Found (%) : C: 54.57;H: 9.65; N: 6.60,
.alpha.].sup.24 .sub.D =-15.49 (1%, water)
.sup.1 H-NMR: 0.88 (3H, t, J=7.2HZ, CH.sub.3 CH.sub.2 CH.sub.2
CH.sub.2 --), 1.16-1.56 (4H, m, CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2
--), 2.17-2.36 (2H, m, CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2 --),
2.48-2.82 (2H, m, H-1a, H-5), 3.02 (1H, dd, J=5.1, 11.4Hz, H-1e),
3.22 (1H, t, J=9.9 Hz, H-1e), 3.66 (1H, t, J=9.4Hz, H-3), 3.44-3.6
(1H, m, H-2), 3.74-3.96 (H.times.2, dd.times.2, H.sub.6,
H.sub.6)
PREPARATION EXAMPLE 2
Moranoline, 5 g, 13 g of n-butyl bromide and 17 g of potassium
carbonate were added to 130 ml of dimethylformamide. The mixture
was reacted at 100.degree. C. for 5 hours. Then, the reaction
mixture was treated in a manner similar to Preparation Example 1 to
give 5.1 g of N-(n-butyl)moranoline. Yield, 75.8%.
PREPARATION EXAMPLE 3
To 100 ml of methanol was added 5 g of moranoline. While stirring
at room temperature, a solution of 20 ml of n-butylaldehyde in 50
ml of methanol having dissolved therein 0.7 g of hydrogen chloride
and 3 g of NaCNCH.sub.3 were added to the mixture. The reaction was
carried out overnight. After completion of the reaction, the
solvent was removed under reduced pressure. The residue was
dissolved in followed by partition with chloroform. The aqueous
phase was passed through a column of 200 ml Diaion SA-11A
(OH.sup.-) type followed by thorough washing with water. The
passing liquid was combined with the washing liquid. The mixture
was passed through a column of 200 ml Dowex 50W.times.28 (H.sup.+)
type. After thoroughly washing, elution was conducted with 1N
ammonia water. After the solvent was distilled off under reduced
pressure, the eluate was crystallized from ethanol.
Recrystallization from ethanol gave 5.1 g of N-(n-butyl)moranoline.
Yield, 75.8%.
Typical examples of the compounds in accordance with the present
invention include the following compounds.
______________________________________ Compound No. 1 Moranoline
Compound No. 2 N-methylmoranoline Compound No. 2a
N-(n-Butyl)moranoline Compound No. 3
N-5-Methoxycarbonylpentylmoranoline tosylate Compound No. 4
N-Hydroxyethylmoranoline Compound No. 5
(N-Methoxycarbonylbutyl)moranoline Compound No. 6 Nojirimycin
bisulfite Compound No. 7 1,4-Bis-(3-moranolino-1-pro- penyl)benzene
dihydrochloride Compound No. 8 N-Hexylmoranoline tosylate Compound
No. 9 N-Isoprenylmoranoline Compound No. 10
N-(2-Hydroxydecyl)moranolinetosylate Compound No. 11
N-10-Carboxydecylmoranoline sodium salt Compound No. 12
N-(3-Phenylpropyl)moranolinetosylate Compound No. 13
N-Benzylmoranoline tosylate Compound No. 14 N-Cinnamylmoranoline
hydrochloride Compound No. 15 N-4-Phenylbutylmoranoline tosylate
Compound No. 16 N-(2-Phenoxyethyl)moranoline Compound No. 17
N-(3-Phenoxypropyl)moranoline tosylate Compound No. 18
N-5-(Phenylpentyl)moranolinetosylate Compound No. 19
N-(2-Cyclopentylethyl)moranoline tosylate Compound No. 20
N-[3-(3-Methoxyethoxyphenyl)-2- butenyl]moranoline Compound No. 21
N,N-Dimethylmoranoline ammonium iodide Compound No. 22
N-Ethylmoranoline Compound No. 23 N-Cinnamylmoranoline Compound No.
24 N-Geranylmoranoline tosylate Compound No. 25
N-(2-Hydroxy-3-phenoxypropyl)morano- line tosylate Compound No. 26
N-Farnesylmoranoline tosylate Compound No. 27
N-10-(N-Methylcarbamoyl)decylmorano- line Compound No. 28
N-(4-Phenyl-3-butenyl)moranoline tosylate Compound No. 29
N-(3-Phenyl-2-methyl-2-propenyl)mor- anoline Compound No. 30
N-(3-o-Chlorophenoxypropyl)moranoline Compound No. 31
N-.gamma.-Methyl-4-bromocinnamyl)morano- line Compound No. 32
N-[4-(3-Fluoro-4-methylphenyl)butyl]- moranoline Compound No. 33
N-(p-Ethoxycinnamyl)moranoline Compound No. 34
N-(p-Isopropoxycinnamyl)moranoline Compound No. 35
N-.gamma.-Methyl-m-methylcinnamyl)morano- line Compound No. 36
N-(4-m-Methoxyphenyl-3-pentenyl)- moranoline Compound No. 37
N-(p-Ethoxycarbonylphenoxyethyl)- moranoline[emiglitate] Compound
No. 38 Castanospermine ______________________________________
In addition to those described above, the compounds in accordance
with the present invention further include the following
compounds.
N-Isobutylmoranoline tosylate
N-Hydroxyethylmoranoline tosylate
N-Aminomoranoline hydrobromide
N-Methoxyethylmoranoline tosylate
N-Methoxyethoxyethylmoranoline tosylate
N-Decylmoranoline tosylate
N-(2-Hydroxyhexadecyl)moranoline tosylate
N-(2-Hydroxy-3-p-tolyloxypropyl)moranoline tosylate
N-(2-Hydroxy-3-p-methoxyphenyloxypropyl)moranoline tosylate
N-(2-Hydroxy-3-p-chlorophenyloxypropyl)moranoline tosylate
N-3-Carbamoylpropylmoranoline
N-Nonylmoranoline tosylate
N-undecylmoranoline tosylate
N-(2-Hydroxytetradecyl)moranoline tosylate
N-(4,4-Diphenyl-3-butenyl)moranoline
N-5-Carboxypentylmoranoline
N-farnesylmoranoline
N-(.gamma.-Methyl-4-chlorocinnamyl)moranoline
N-(.gamma.-Methyl-4-methylcinnamyl)moranoline
N-(4-p-Chlorophenyl-3-pentenyl)moranoline
N-(4-m-Chlorophenyl-3-pentenyl)moranoline
N-(4-o-Chlorophenyl-3-pentenyl)moranoline
N-(4-p-Phenoxyphenyl-3-pentenyl)moranoline
N-(4-p-Ethoxyphenyl-3-pentenyl)moranoline
N-(m-Methoxycinnamyl)moranoline
N-[3-(3-Chlorophenyl)-2-butenyl]moranoline
N-[4-(4-Chlorophenyl)-3-butenyl]moranoline
N-(4-Carboxylcinnamyl)moranoline hydrochloride
N-(3-Carboxy-2-propenyl)moranoline
N-(m-Triethylammonioethoxycinnamyl)moranoline dipicrate
N-Isopropylmoranoline
N-(p-Trimethylammonioethyoxycinnamyl)moranolinechloride
hydrochloride
When the compounds of the present invention are administered as
pharmaceuticals, to humans and animals, they are given per se or as
a pharmaceutical composition containing, for example, 0.1 to 99.5%
(more preferably, 0.5 to 90%) of active ingredient in combination
with a pharmaceutically acceptable carrier.
The compounds of the present invention may be given orally,
parenterally, topically, or rectally. They are of course given by
forms suitable for each administration route. For example, they are
administered in tablets or capsule form, by injection, inhalation,
eye lotion, ointment, suppository, etc. administration by
injection, infusion or inhilation; topical by lotion or ointment;
and rectal by suppositories. Oral administration is preferred.
As to carriers, one or more liquid, solid or semisolid diluent,
filler and other auxillary agents for pharmaceutical preparations
may be used. It is desired that the pharmaceutical compositions are
administered in unit dosage form.
Oral administration can be effected utilizing solid and liquid
dosage unit forms such as powders, tablets, capsules, granules and
the like.
Powders are prepared by comminuting the compound to a suitable fine
size and mixing with a similarly comminuted pharmaceutical carrier
such as an edible carbohydrate as, for example, starch or mannitol.
Flavoring, preservative, dispersing and coloring agents can also be
present.
Capsules are made by preparing a powder mixture as described above
and filling formed gelatin sheaths. Glidants and lubricants such as
colloidal silica, talc, magnesium stearate, calcium stearate or
solid polyethylene glycol can be added to the powder mixture before
the filling operation. A disintegrating or solubilizing agent such
as agar-agar, calcium carbonate or sodium carbonate can also be
added to improve the availability of the medicament when the
capsule is ingested.
Tablets are formulated, for example, by preparing a powder mixture,
granulating or slugging, adding a lubricant and disintegrant and
pressing into tablets. A powder mixture is prepared by mixing the
compound, suitably comminuted, with a diluent or base as described
above, and optionally, with a binder as carboxymethyl cellulose, an
alginage, gelatin, or polyvinyl pyrrolidone, a solution retardant
such as paraffin, a resorption accelerator such as a quarternary
salt and/or an absorption agent such as bentonite, kaolin or
dicalcium phosphate. The powder mixture can be granulated by
wetting with a binder such as syrup, starch paste, acadia mucilage
or solutions of cellulosic or polymeric materials and forcing
through a screen. As an alternative to granulating, the powder
mixture can be run through the tablet machine and the resulting
imperfectly formed slugs broken into granules. The granules can be
lubricated to prevent sticking to the tablet forming dies by means
of the addition of stearic acid, a stearate salt, talc or mineral
oil. The lubricated mixture is then compressed into tablets. The
compounds and pharmaceutically accetable acid addition salts of the
present invention can also be combined with free flowing inert
carriers and compressed into tablets directly without going through
the granulating or slugging steps. A clear or opaque protective
coating consisting of a sealing coat of shellac, a coating of sugar
or polymeric material and a polish coating of wax can be provided.
Dyestuffs can be added to these coatings to distinguish different
unit dosages.
Oral fluids such as solutions, syrups and elixirs can be prepared
in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a nontoxic alcoholic
vehicle. Suspensions can be formulated by dispersing the compound
in a nontoxic vehicle. Solubilizers and emulsifiers such as
ethoxylated isostearyl alcohols and polyoxyethylene sorbitol
esters, preservatives, flavor additives such as peppermint oil or
saccharin, and the like can also be added.
Where appropriate, dosage unit formulations or oral administration
can be microencapsulated. The formulation can also be prepared to
prolong or sustain the release as for example by coating or
embedding particulate material in polymers, wax or the like.
Parenteral administration can be effected utilizing liquid dosage
unit forms such as sterile solutions and suspensions intended for
subcutaneous, intramuscular or intravenous injection. These are
prepared by suspending or dissolving a measured amount of the
compound in a nontoxic liquid vehicle suitable for injection such
as aqueous or oleaginous medium and sterilizing the suspension or
solution. Alternatively, a measured amount of the compound is
placed in a vial and the vial and its contents are sterilized and
sealed. An accompanying vial or vehicle can be provided for mixing
prior to administration. Nontoxic salts and salt solutions can be
added to render the injection isotonic. Stabilizers, preservatives
and emulsifiers can also be added.
Rectal administration can be effected utilizing suppositories in
which the compound is admixed with low-melting, water-soluble or
insoluble solids such as polyethylene glycol, cocoa butter, higher
esters as for example flavored aqueous solution, while elixirs are
prepared through myristyl palmitate or mixtures thereof.
In determining the specific dosage for each treatment, a number of
factors such as the age of the patient, body weight, severity of
condition, administration route, and the like must be considered.
Generally, from about 50 mg to 3000 mg per day of a compound of the
present invention should be administered to a human adult
preferably from 500 mg to 1000 mg per day.
It is preferred that the administration be divided so that
administration takes place 2 or 3 times per day.
The following nonlimitative examples more particularly illustrate
the present invention.
The .alpha..sub.2 -PI lowering activity and the urokinase secretion
accelerating activity, as well as, the low toxicity of the
compounds of the present invention are illustrated by the data set
forth below.
Activity in vitro
It is known that human hepatic cancer-derived HepG2 cells
synthesize and secrete .alpha..sub.2 -plasmin inhibitor
(.alpha..sub.2 -PI). The HepG2 cells, 2.times.10.sup.6, were
inoculated on a plastic culture plate (diameter of 100 mm) made by
Falcon Inc. and cultured in Eagle's minimum medium containing 10%
bovine fetal serum.
Three days after, cells adhered to the bottom of the plate were
washed twice with Dulbecco's phosphate buffer followed by culturing
in 8 ml of serum-free Eagle's medium (containing no Phenol Red)
containing 200 .mu.g/ml of a specimen of the compound of the
present invention for further 3 to 4 days. After culture, 7 ml of
the medium was collected and concentrated to about 1 ml using
Centriflow (CF25) made by Falcon Inc. The concentrate was further
freeze dried.
To the freeze dried sample, 0.7 ml of 50 mM Tris buffer containing
8.1 mg/ml of monomethylamine hydrochloride was added to dissolve
therein, whereby the solution was concentrated to 10-fold.
To 100 .mu.l of the concentrated sample, 50 .mu.l of 15 mCU plasmin
solution was added and further 50 .mu.l of 0.25 .mu.mole S-2251
synthetic chromogenic substrate solution was added thereto followed
by reacting at 37.C for 10 minutes. By adding 1 ml of 2% citrate
solution, the reaction was stopped. p-Nitroanilide released from
the S-2251 substrate was measured at O.D. of 403 nm.
A sample in which decomposition of the S-2251 substrate was
measured using the aforesaid Tris buffer instead of the
concentrated sample was made a control showing 100% plasmin
activity and the concentrated sample cultured without adding a test
compound was made a control showing 100% .alpha..sub.2 -PI
acitivity. The results were calculated according to the following
equation. ##EQU1## wherein A.sub.1, A.sub.2 and A.sub.3 represent
absorbance in 100% control, absorbance when the concentrated sample
was used and absorbance in a .alpha..sub.2 -PI activity 100%
control, respectively. The number of the samples was 3,
respectively. The results are shown in Table 1. It is evidence that
the compounds of the present invention can lower the .alpha..sub.2
-PI activity.
TABLE 1 ______________________________________ Sample .alpha..sub.2
-PI Activity Sample .alpha..sub.2 -PI Activ- Compound No. (%)
Compound No. ity (%) ______________________________________ Control
100 11 88 1 63 12 50 2 62 13 80 2a 59 3 70 14 51 4 70 15 39 5 79 16
74 6 67 17 65 7 85 18 20 8 56 19 63 9 55 20 37
______________________________________
Activity in vivo
Three (3) male Beagle dogs were used for the control group and the
administered group, respectively, as animals to be
administered.
A test sample (Compound No. 2) was dissolved in a concentration of
10 mg 0.1 ml, using 0.1 M phosphage buffer (pH 7.2). After
dissolution, the solution was sterilized by filtration through a
sterile filter (pore size, 0.2 .mu.m) and then 0.3 ml of the
solution was administered per 1 kg of body weight (30 mg/kg). The
administration was made through the right front limb vein for
consecutive 7 days. Collected blood was mixed with 3.8% sodium
citrate in a ratio of 1:9 by volume. By centrifugation at 3000 rpm
for 15 minutes, plasma was isolated.
The .alpha..sub.2 -PI activity was measured as an inhibition
activity against decomposition of plasmin with the synthetic
chromogenic substrate S-2251. The results of measurement are shown
as change in inhibition activity after administration based on 100%
of the plasmin inhibition activity prior to administration of the
test sample (Table 2). The sample number in each group was 3
samples.
The .alpha..sub.2 -PI activity was obviously depressed by
consecutive administration of the test compound in a dose of 30
mg/kg.
TABLE 2 ______________________________________ [.alpha..sub.2 -PI
activity (%)] Day 0 1 2 3 4 5 6 7
______________________________________ C 100 98 92 94 96 98 100 106
Sample 100 95 83 84 76 75 86 89
______________________________________ C: control Sample: test
sample
Test on fibrinolysis in vitro
A test sample (Compound No. 2) was consecutively administered to
Beagle dogs in a dose of 30 mg/kg once a day. Plasma was isolated
prior to and 4 days after the administration. The .alpha..sub.2 -PI
activity in plasma was measured and at the same time, fibrin clot
was formed in vitro using the plasma. When urokinase as a
thrombolytic agent was acted on the fibrin clot, a degree of lysis
was compared between plasma prior to the administration and plasma
4 days after the adminsitration.
To 500 .mu.l of the isolated plasma, 40 .mu.l of .sup.125
I-fibrinogen (0.1 mCi/ml) was added and 50 .mu.l each of the
mixture was dispensed in a test tube. 5 .mu.l each of a solution
mixture of 25 U/ml thrombin and 0.5 M calcium chloride was added to
each test tube, which was incubated at 37.degree. C. for 30 minutes
to prepare fibrin clots. Solutions of 15 and 30 U/ml of urokinase
in 2% albumin solution and the resulting solution were charged in
each test tube by 1 ml each.
After incubating at 37.degree. C. for 12 hours, 25 .mu.l of the
supernatant was collected in an RAI tube and .sup.125 I-fibrin
decomposition products isolated in the supernatant were measured
with a y-counter. The sample number was 3 in each group.
As shown in Table 3, it is clear that when the plasma having
lowered .mu.2-PI activity was used, lysis of the fibrin clots was
accelerated by administration of the test compound, as compared to
the plasma prior to the administration.
TABLE 3 ______________________________________ Rate of Lysis of
Fibrin Clot (%) Urokinase Prior to 4 Days After Activity
Administration Administration
______________________________________ 0 U 18.1 21.5 15 U 37.4 63.8
30 U 70.0 93.7 ______________________________________
Thrombolytic test in vitro
Thrombolytic activity-inducing ability in the established vascular
endothelial cell culture system was examined using CPAE (calf
pulmonary artery endothelia).
CPAE cells were purchased from Dainippon Pharmaceutical Co., Ltd.,
Department of Laboratory Products. CPAE cells were subcultured in
10% FCS-Eagle MEM medium charged in a culture flask of 25 om.sup.2.
From the cell suspension fractioned upon subculture, 0.1 ml was
transferred into a test tube with a sterilized pipette. Then, the
cell suspension was diluted to 10 fold with 0.9 ml of Trypan Blue
solution and a cell count was counted with a cell counter. After
diluting with 10% FCS-Eagle MEM medium so as to have a cell count
of 2.times.10.sup.5 cells/ml, 100 .mu.l each/wall, namely,
2.times.10.sup.4 cells/wall, of the dilution was dispensed in a 96
wall microtiter plate (manufactured by Corning) with a
micropipette. The plate was incubated at 37.degree. C. in 5%
CO.sub.2.
The compound of the present invention was dissolved in medium in
0.2 mg/ml; the compound that was insoluble in the medium was
dissolved in less than 1% of DMSO and the resulting solution was
aseptically filtered through a filter; 5 .mu.l of the solution was
added to the wall with a sterilized micropipette 24 hours after
onset of the incubation. After culturing at 37.degree. C. for 72
hours in 5% CO.sub.2, the collected culture supernatant was
provided for measurement.
The measurement was performed as follows. Plasminogen, 5 .mu.l, was
charged in a wall of fibrin plate (manufactured by Kitazato
Institute), which was allowed to stand until diffusion was
completed. After diffusion, 5 .mu.l of the supernatant was charged
and then put in a carbon dioxide gas incubator of 37.degree. C.
Four hours after, evaluation was made by formation of a transparent
lysis circle by fibrinolysis. In this case, it was confirmed that a
transparent circle was simultaneously formed by fibrinolysis in a
wall charged with t-PA as a positive control. A diameter of the
transparent circle was 9 to 10 mm.
Also in the case of the supernatant added with the compound of the
present invention, a transparent circle due to fibrinolysis showing
a diameter of 4.5 to 8.5 mm was noted. However, in the case of the
supernatant added with no compound of the present invention
(control), no change was noted. In the case of forming a circle of
4 mm or more due to fibrinolysis, it was judged that thrombolytic
ability of CPAE cells was induced.
After the fibrinolytic activity was checked over, the wall was
fixed with 2.5% of glutaraldehyde in a final concentration. Then,
the solution was discarded and the system was washed with PBS.
After washing, moisture was removed. After staining with 100 .mu.l
of 0.1% crystal violet and allowing stand for 2 to 3 minutes, the
system was washed with running water. After an excess of the
staining solution was washed out, moisture was removed and the dye
bound to the cells was eluted by 100 .mu.l of methanol. Using
multiscanning (Titertech), measurements were performed at a
wavelength of 580 nm in accordance with the ABS method and the
matrix method to confirm that the cells were not injured.
A diameter (mm) of the circle due to fibrinolysis is shown in Table
4. It is clearly seen that the compounds of the present invention
showed the thrombolytic activity.
TABLE 4 ______________________________________ Diameter Compound
No. Diameter (mm) Compound No. (mm)
______________________________________ 2 7.9 25 4.1 8 6.3 26 5.7 9
8.2 27 8.5 10 4.5 28 8.3 12 5.9 29 7.1 14 5.9 30 7.6 15 6.3 31 7.9
17 7.5 32 7.5 18 6.8 33 7.8 19 5.8 34 7.6 20 8.0 35 7.8 21 5.6 36
7.6 22 6.5 37 6.9 23 8.4 38 6.9 24 7.6 2a 7.5
______________________________________
Thrombolytic activity in vitro
The compounds of the present invention have been shown to be
capable of inducing fibrinolytic activity in CPAE cells. In order
to verify by what substance in addition to the .alpha..sub.2 -PI
lowering activity, this fibrinolytic activity is induced, analysis
was made on the culture solution added with Compound No. 2 out of
the compounds of the present invention, by means of fibrin
autography.
By SDS-polyacrylamide gel using 10% gel, this culture solution and
t-PA (tissue plasminogen activator) and urokinase were subjected to
electrophoresis. After the electrophoresis, the gel was treated
with 2.5% Triton x-100, which was inoculated on agar plate added
with fibrinogen, thrombin and plasminogen. A place of fibrin which
caused lysis was confirmed in an incubator of 37.degree. C. in 5%
CO.sub.2.
As a result, great fibrinolysis was noted in the culture
supernatant added with the compound of the present invention, at
the position of molecular weight of urokinase type plasminogen
activator. It was made clear that the compound of the present
invention strongly induced production of urokinase type plasminogen
activator in CPAE.
Test on acute toxicity
Four (4) ddY strain male mice of 6 week age were used for each
group of the test sample.
Method
In intravenous administration, each sample was dissolved in 0.9%
physiological saline and the solution was administered through the
tail vein. In intraperiotoneal administration, each sample was
suspended in 0.5% CMC-physiological saline and 0.1 ml of the
suspension per 10 mg of mouse body weight was intraperitoneally
administered. In oral administration, each sample was suspended in
0.5% CMC-physiological saline and 0.2 ml of the suspension per 10
mg of mouse body weight was orally administered.
Observation was made immediately after the administration. After
observation for 1 week after the administration, the mice was
sacrificed with chloroform and subjected to autopsy.
LD.sub.50 of each sample is summarized in the following table.
Safety of the compounds of the present invention are clearly
shown.
______________________________________ (Compound No.) LD.sub.50
(mg/kg) ______________________________________ Intravenous
administration: 1 3235 2 5091 Intraperitoneal administration: 1
5,000 2 10,000 Oral administration: 1 7,500 2 10,000
______________________________________
The following examples illustrate the formulation of pharmaceutical
compositions according to the present invention:
EXAMPLE 1
Per 1 tablet, the following compounds were added to the compound
(Compound No. 2) of the present invention and tablets were obtained
in a conventional manner.
______________________________________ Per tablet (in 300 mg) 200
mg Compound of the present invention (Compound No. 2) Lactose 50 mg
Corn starch 20 mg Low substitution degree 15 mg hydroxypropyl
cellulose Hydroxypropyl cellulose 5 mg Magnesium stearate 10 mg 300
mg ______________________________________
EXAMPLE 2
Per 1 ampoule, the following compounds were added to the compound
(Compound No. 2) of the present invention and ampoules for
injection were obtained in a conventional manner.
______________________________________ Per ampoule (in 10 ml) 200
mg Compound of the present invention (Compound No. 2) Sodium
chloride 90 mg Distilled water for injection c.s. 10 ml
______________________________________
* * * * *